There is a large body of evidence suggesting that oxidative modifications of low-density lipoproteins (LDL) contribute significantly to the initiation and/or progression of atherosclerosis. Although numerous studies of the oxidation of LDL lipids have been published, far less is known about the oxidative modification of the apoprotein. The greater chemical diversity of the apoprotein than of the LDL lipids offers the opportunity for greater biomarker specificity for distinguishing contributions of specific mechanisms of oxidation. The first Specific Aim of the present proposal is to test the hypothesis that oxidations of LDL in vitro by methods that are candidate mechanisms for oxidation of LDL in vivo produce modifications of the apoprotein (apo B-100) that are sufficiently distinguishable to be empIoyed as biomarkers for oxidation of LDL in vivo by the respective mechanisms. In our studies to date we have observed clear differences in products of apoB-1 00 oxidation formed in oxidation of LDL in vitro by Cu2+, HOCl, and myeloperoxidase (MPO). We propose to investigate similarly the oxidation of LDL in vitro by Fe2+-catalyzed oxidation, nitration (NO and ONOOdonors, MPO or eosinophil peroxidase aboutPO] plus NO2-), and oxidation mediated by cultured cells, including endothelial cells, vascular smooth muscle cells and monocytes. These studies will employ isolation methods based on high performance liquid chromatography (HPLC) and structural characterization methods based on mass spectrometry. The second Specific Aim of this proposal is to test the hypothesis that subfractions of circulating LDL isolated from some individuals will exhibit specific modifications apoB-1 00 oxidation that will reflect a limited subset of the products of oxidation of LDL in vitro characterized in Specific Aim 1. The third Specific Aim of this proposal is to test the hypothesis that LDL isolated from atheromatous material will exhibit a limited subset of the products of apoprotein oxidation that were characterized in Specific Aim i. Our results to date provide very strong support for the working hypotheses upon which we base this application. The goal of the studies proposed is to develop a means of distinguishing the specific mechanisms of LDL oxidation that are suspected to contribute to the oxidation of LDL in vivo and to atherogenesis. The longer-term goals of this research are to create the basis for more mechanistically specific therapeutic efforts to retard the initiation and progression of atherosclerosis and to develop biomarkers of these distinct mechanisms of oxidation to monitor efficacies of these therapeutic interventions. Finally, the studies we propose are not limited in relevance to atherosclerosis or even to oxidation of LDL, but the concepts and methods developed in these studies are readily applicable to a wide range of important human diseases for which unspecified oxidative processes are proposed to contribute.